This application relates to hybrid cell lines (lymphocyte hybridomas) for the production of monoclonal antibodies to xcex1vxcex23 integrin, to such homogeneous antibodies, and to the use of such antibodies for diagnostic and therapeutic purposes.
xcex1vxcex23 is a member of the integrin supergene family of cell-surface lycoprotein receptors that promote cellular adhesion. Each cell has a specific repertoire of receptors that define its adhesive capabilities. The integrins are expressed as heterodimers of noncovalently associated xcex1 and xcex2 subunits. According to the nomenclature proposed by Hynes, R. O. [Cell 48, 875-886 (1987)], the integrins can be divided into families each with a common xcex2-subunit and a set of variable xcex1-subunits known to associate with the common xcex2-subunit. The different xcex1 chains are denoted by the original cell type, by a subscript used by the original discoverer, or, as in the case of the xcex1vxcex23 receptor, by the nature of the ligand (i.e. xcex1v stands for a vitronectin receptor xcex1-chain). Many, but not all, integrin receptors have been shown to interact with proteins via a tripeptide sequence, Arg-Gly-Asp (or RGD using the single letter amino acid code), originally defined from studies of the cell binding domains of fibronectin [Ruoslahti, E. and Pierschbachter, M. D., Cell 44, 517-518 (1986); Ruoslahti, E. and Pierschbachter, M. D., Schience 238, 491-497 (1987)].
xcex1vxcex23 (also referred to as vitronectin receptor or VNR) is a member of the xcex23 integrin subfamily and is expressed on a variety of cells, including endothelial, melanoma, smooth muscle cells and, along with another integrin xcex12xcex21 (VLA-2) (the receptor for Type I collagen and laminin), on the surface of osteoclasts [Horton, M. A. and Davies, J., J. Bone Min. Res. 4, 803-808 (1989); Davies, J. et al., J. Cell. Biol. 109, 1817-1826(1989); Horton, M., Ing. J. Exp. Pathol. 71 741-759 (1990)]. xcex1vxcex23 mediates cell adhesion to vitronectin, fibrinogen, fibronectin, thrombospondin, osteopontin, bone sialo protein II and von Willebrand factor.
Osteoclasts are the main type of bone cells involved in the resorption of bone tissues. The resorption process involves the proliferation and chemotaxis of developing osteoclasts to the skeleton from hematopoietic sites, migration of mature cells to sites of subsequent resorption, attachment of osteoclasts to bone substrate and the eventual formation of the polarized, functional mature end cells which are directly involved in bone resorption. The xcex1vxcex23 integrin mediates adhesion of osteoclasts to RGD sequence-containing bone matrix proteins.
Antibodies to xcex1vxcex23 are expected to be valuable diagnostic and therapeutic tools in studying the biological role and the structural/functional relationships of this integrin with its various ligands. In particular, monoclonal antibodies (Mabs) detecting unique epitopes on osteoclasts would be of great value in understanding of the development of osteoclasts. Even more importantly, neutralizing Mabs specific for xcex1vxcex23 that inhibit the osteoclast binding to the bone matrix proteins have great potential as therapeutic agents useful in the treatment of conditions associated with excessive bone resorption.
There are several monoclonal antibodies known in the art that bind to various epitopes on xcex1vxcex23. Immunizing with osteoclasts from osteoclastomas (giant cell tumors of bone), Horton, M. A. et al. [Cancer Res. 45, 5663-5669 (1985)] produced eleven mouse hybridomas secreting monoclonal antibodies which bind to osteoclasts in normal human fetal bone and a variety of neoplastic and non-neoplastic bone lesions. One of these, designated 23C6, was subsequently shown to bind the xcex1vxcex23 complex, and was demonstrated to be able to disrupt osteoclast function [Horton, M. A. et al., Exp. Cell. Res. 195 368-375 (1991)]. Another monoclonal antibody, LM609 (produced in hybridoma LM609 ATCC HB 9537) disclosed in PCT Application Publication No. WO 89/05155 (published Jun. 15, 1989) and Cheresh et al. J. Biol. Chem. 262: 17703-17711 (1987) was also found to bind the xcex1vxcex23 complex and, due to its ability to inhibit the binding of ECr molecules present on the surface of tumor cells and blood vessel forming endothelial cells to vitronectin, fibrinogen and von Willebrand factor, was proposed for therapeutic use as tumor growth inhibitor. Monoclonal antibody 13C2(Horton, M. A. et al., Cancel Res. 1985, Supra) was shown to bind the xcex1v portion of the xcex1vxcex23 molecule, whereas several other monoclonal antibodies were reported to recognize the xcex23 portion [Nesbitt, S. et al., Epitope Analysis of the Vitronectin Receptor (CD51), In xe2x80x9cLeukocyte Typing IVxe2x80x9d White Cell Differentiation Antigens, Knapp, W. et al. (eds.) 1991, p. 1037]. The specific monoclonal antibodies variously reported in the art were shown to also bind to endothelial cells and various melanoma cell lines.
There is a need for high affinity monoclonal antibodies to the xcex1vxcex23 integrin that are capable of effective inhibition of the binding of xcex1vxcex23 expressing cells to xcex1vxcex23 ligands, such as vitronectin and fibronectin.
It would be further desirable to provide monoclonal antibodies to xcex1vxcex23 that bind osteoclasts and optionally other cells known to express xcex1vxcex23.
It would be particularly desirable to provide monoclonal antibodies that are effective inhibitors of xcex1vxcex23 binding to its ligands and which specifically bind osteoclasts without binding to other cells known to express xcex1vxcex23, i.e., which are more specific for the target integrin on osteoclasts.
The present invention is based on successful research involving the production and extensive characterization of monoclonal antibodies to xcex1vxcex23 integrin. Accordingly, the present invention is directed to monoclonal antibodies, and derivatives thereof, which are capable of recognizing unique epitopes on xcex1vxcex23 and/or which exhibit high affinity for xcex1vxcex23. The invention is specifically directed to monoclonal antibodies recognizing unique epitopes on the xcex1vxcex23 complex or the xcex23 portion thereof. The invention is further directed to monoclonal antibodies effectively inhibiting the binding to vitronectin and fibrinogen of xcex1vxcex23 expressing cells. In a particularly important aspect, the invention is directed to monoclonal antibodies specifically binding xcex1vxcex23 on osteoclasts but not other xcex1vxcex23 on other cells (e.g. melanoma cells C32R, M-21, HA-A, HA-L and HT-144 and human umbilical vein endothelial cells).
In one aspect, the invention concerns an anti-xcex1vxcex23 monoclonal antibody that is capable of: (1) inhibiting the binding of xcex1vxcex23 expressing cells to fibrinogen, (2) binding osteoclasts, and (3) binding to substantially the same epitope recognized by any one of a monoclonal antibody selected from the group consisting of 10C4.1.3, 9G2.1.3 and 9D4.9.1 or which has an affinity for xcex1vxcex23 which is about equal to or greater than that of the foregoing three antibodies.
In another aspect, the invention concerns isolated nucleic acid encoding such antibodies, and hybridoma or recombinant cells producing such antibodies.
In a further aspect, the invention concerns the therapeutic or diagnostic use of such antibodies. The monoclonal antibodies of the invention are useful as therapeutic agents, either by themselves or in conjunction with (chemo)therapeutic agents, to treat diseases or conditions that are characterized by excessive bone resorption and/or to inhibit tumor growth. The monoclonal antibodies of the invention are also useful in diagnostic and analytical assays for determining the presence of xcex1vxcex23 on cells, cell typing and in histochemical tissue staining.
These and further aspects will be apparent from the following detailed description.